Download THE EFFECT OF VARIOUS ACIDS ON THE DIGESTION OF

Published July 20, 1919
T H E E F F E C T OF VARIOUS ACIDS ON T H E DIGESTION OF
PROTEINS BY PEPSIN.
BY J. H. NORTHROP.
(From the Laboratories of The Rockefeller Institute .for Medical Research.)
(Received for publication, May 21, 1919.)
I.
x Falk, I. S., Y. Biol. Chem., 1918, xxxvi, 229.
2 Loeb, J., y. Biol. Chem., 1918, xxxiii, 531; J. Gen. Physiol., 1918-19, i, 39, 363,
483,559.
3 Loeb, J., J. Gen. Physiol., 1918-19, i, 237.
4 Pfleiderer, R., Arch. ges. Physiol., 1897, lxvi, 605. A review of the early
literature is given in this article.
5 Berg, W. N., and Gies, W. J., J. Biol. Chem., 1906--07, ii, 489.
6O7
The Journal of General Physiology
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The widespread occurrence of antagonistic salt action on living
tissues raises the question whether the underlying cause of the phenomenon might not be found in the action of the salts on the activity
of the enzymes. A similar effect has been described by Falk ~ in the
case of lipase. As Loeb ~,3 has shown, it is also possible to demonstrate
antagonistic salt action on the physical properties of a protein; i.e.,
gelatin. I t seemed important therefore to determine whether or not
such a n effect was to be found in the influence of various acids on the
digestion of proteins by pepsin•
The relative action of the various acids on the pepsin digestion of
proteins has already been the subject of many investigations. 4 The
literature on the subject is confused and contradictory, however, due
largely to the fact that in the early work the effect of the hydrogen
ion concentration was not taken into account and that the acids
were usually compared in equimolecular or even equipercentage
strengths. Attempts were made by Berg and Gies5 to rule out this
disturbing factor by using solutions containing equal calculated
amounts of hydrogen ion. They took no account, however, of the
"buffer" action of the weaker acids and of the proteins so that their
Published July 20, 1919
608
E F F E C T OF ACIDS ON DIGESTION OF PROTEINS
S6rensen, S. P. L., Biochem. Z., 19(i9, xxi, 131.
Michaelis, L., and Mendelssohn, A., Biochem. Z., 1914, lxv, 1.
s Ringer, W. E., Kolloid-Z., 1916, xix, 253; Arch. n~erl, de Physiol., abstracted
in Physiol. Abstr., 1919, iii, 408.
9Van Slyke, D. D., J. Biol. Chem., 1913-14, xvi, 121.
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solutions probably did not have the same hydrogen ion concentration.
S6rensen ~ has shown that the activity of pepsin depends very largely
on the hydrogen ion concentration, and at the same time has developed accurate methods for the determination of the latter. In
view of his work it is obvious that the comparison of the action of
the different acids must be made in such a way as to keep this factor
constant. Failure by the earlier workers to do this probably accounts
in large measure for the great differences in the observed efficiency
of the various acids.
More recently Michaelis and Mendelssohn 7 have shown that the
optimum acidity for the digestion of edestin by pepsin is the same for
hydrochloric, nitric, tartaric, and oxalic acids. Ringer 8 states that
the optimum reaction depends on the protein used and coincides
with the maximum hydration of the protein as measured by the viscosity. A somewhat similar theory was proposed by Pfleiderer 4 who
attempted to show a relation between the rate of digestion of fibrin
and the amount of swelling in various acids.
In all these investigations the amount of digestion was followed
by determining (1) the amount of solution of an insoluble substrate,
(2) the amount of precipitable protein left in solution, or (3) the
rate of liberation of carmine from carmine fibrin. As has been
pointed out by various authors, there is considerable doubt as to
whether any of these methods actually follows the chemical changes
in the structure of the protein during hydrolysis.
The recent improvements in the technique of the determination
of amino nitrogen by the Van Slyke 9 method make it possible to follow
the increase in the number of free amino groups. From our knowledge
of the changes involved in the hydrolysis of proteins it would seem
that this increase probably follows accurately the amount of hydrolysis. The changes are small, however, and even with the greatest
care it is difficult to get strictly accurate figures.
Published July 20, 1919
J, H. NOP,THROP
609
In the present work the rate of pepsin digestion of gelatin, egg
albumin, edestin, blood albumin, and casein in the presence of hydrochloric, nitric, acetic, sulfuric, oxalic, phosphoric, and citric
acids has been followed by this method. The determinations were
made at two ranges of hydrogen ion concentration, pH 1.0 to 1.5 and
pH 2.5 to 3.5.
A summary of the results obtained with edestin at a reaction of
pH 2.6 is given in Table I. The results obtained with the other proTABLE I.
Quantity of Amino Nitrogen per 10 cc. of Solution at 24°C. and 750 ram.
Substrate, 20 cc. of edestin solution A. ~ ~. ,
Pepsin,
20 cc. of solution A.
f r m a t volume, 120 cc.
Acid.
Time.
HC1
HNO3
! etlc
H~SO4
6.
CG,
Oxalic
C.
Citric
HCI*
CC.
CG.
h~'S.
CO,
0
0
O. 50
0.50
0.49
0.50
.49
.48
0.48
0.49
0,51
0,48
.50
.51
0.49
0.48
0.49
0.49
4
4
1:28
1.25
1.23
1,21
.09
1.33
1.25
1.22
1.26
.24
.25
1.20
1.24
0.50
.16
24
24
1.55
1.51
1.58
1.50
.17
.37
1.56
1.48
1.48
1.50
.44
,52
1.39
1.49
0,51
0,50
2.3
2.3
0,47
pH of solution.
2
24
2.5
2.7
2.6
2.4
2.7
2.4
* Control with boiled pepsin solution.
teins were practically identical with these and therefore will not be
given here. The experiraents show that the rate of hydrolysis of all
the proteins studied is identical, for all the acids (except acetic)
within the rather wide range of error of the method used (about 5
per cent). With gelatin acetic acid also behaves quantitatively like
the other acids even in concentrations as high as 25 volumes per
cent. With the other proteins the rate of hydrolysis in the presence
of acetic acid is slower than with HC1, HNO3, H~SO4, oxalic, citric,
or phosphoric acids. The effect therefore is evidently on the protein
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Experiment
No.
Published July 20, 1919
610
E F F E C T O F ACIDS O N DIGESTION O F P R O T E I N S
and not on the enzyme. These experiments also show that the physical properties of the solution, such as viscosity, have little or no
effect on the rate of digestion since Loeb has shown that there is a
striking difference in the viscosity of gelatin in sulfuric as contrasted
with hydrochloric acid solution. This is still more strikingly shown
in the experiments with edestin, which is practically insoluble in
sulfuric acid and yet digests under these conditions at the same rate
as when dissolved in hydrochloric acid. The simplest explanation of
these results would seem to be that the rate of digestion of the protein is determined by the amount of acid protein salt formed. As
Loeb ~,3 has shown, the physical properties of a gelatin solution are
also functions of this same quantity.
II. EXPERIMENTAL.
bottles containing the digestion mixtures at intervals of 4 and 24
hours and placed in iced bottles containing three drops of saturated
ferric chloride solution. They were kept at 2--4°C. until analyzed.
No change could be noted in the amino nitrogen content in 24 hours
under these conditions.
Temperature.--Some of the experiments were conducted at 37 ° 4- 0.1,
and some at 3504 - 0.1.
Analysis.--The technique was the same as described by Van Slyke
except that 10 cc. of solution were analyzed in a large size apparatus
and the gas was measured in a small (3 cc.) burette. This was
necessary owing to the small amount of amino nitrogen present.
The protein solution was allowed to stand 15 minutes in contact
with the nitrous acid and then shaken rapidly for 5 minutes. The
reaction was complete under these conditions.
Pepsin Soluaon A.--30 gm. of Fairchilds pepsin were dissolved in
500 cc. of water and allowed to digest at 37 ° for 24 hours. The solution was dialyzed and filtered, and made up to 3 liters. It contained
0.1 cc. of amino nitrogen per 10 cc. This quantity did not further increase digestion in 24 hours and therefore does not enter into the
figures obtained for the increase of amino nitrogen in the solutions.
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Method of Keeping Samples.--Samples were withdrawn from the
Published July 20, 1919
j. 1~. ~OlZrSmoP
611
It was found t h a t 20 cc. of this solution added to 100 cc. of the
protein solution caused the hydrolysis to be about one-third complete
in 4 hours and two-thirds complete in 24 hours. This concentration
was therefore used.
Edestin Solution A.--25 gm. of crystalline edestin were dissolved
in 300 cc. of dilute NaOH and precipitated by the addition of dilute
HCL. The reaction was adjusted to the isoelectric point of edestin
and the solution then dialyzed for a week against tap water and 2
days against distilled water. It was then diluted to 500 cc. A fine
suspension was obtained which could be accurately pipetted. The
conductivity was about that of an M/l,500 KCL solution, showing that
only traces of electrolytes were present. The other protein solutions were purified in the same way by dialysis at the isoelectric
point (Loeb)?
The required amount of protein solution was pipetted into a 100
cc. volumetric flask and a drop of indicator added (methyl orange or
thymol blue, depend{ng on the reaction desired). Hydrochloric acid
was then added until the approximate reaction desired was reached.
The solutions containing the other acids were prepared in the same
way by adding the acid to the protein solution until the color matched
exactly that of the flask containing the hydrochloric acid solution.
In this way solutions of the same pH could be easily prepared.
Control experiments showed that the indicator had no effect on
either the rate of digestion or the analysis. The absolute reaction
of the mixtures could not be measured colorimetrically owing to the
"protein error." A sample of the solution was removed shortly
after adding the pepsin therefore and the pH determined by the
E.~t.F. method. It was found as stated by S6rensen that the change
in reaction during the digestion was insignificant.
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Adjustment and Measurement of the Reaction.
Published July 20, 1919
612
EFFECT
OF
ACIDS
ON
DIGESTION
OF
PI~OTEII~S
SUMM_AlZY.
1. At equal hydrogen ion concentration the rate of pepsin digestion
of gelatin, egg albumin, blood albumin, casein, and edestin is the
same in solutions of hydrochloric, nitric, sulfuric, oxalic, citric, and
phosphoric acids. Acetic acid diminishes the rate of digestion of all
the proteins except gelatin.
2. There is no evidence of antagonistic salt action in the effect of
acids on the pepsin digestion of proteins.
3. The state of aggregation of the protein, i.e. whether in solution
or not, and the viscosity of the solution have no marked influence on
the rate of digestion of the protein.
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The author is indebted to Drs. Van Slyke and Cullen for placing
their apparatus for the hydrogen ion determination at his disposal.